Sphingolipids segregate into nano-scaled microdomains at the cellular plasma membrane, commonly referred to as lipid rafts, which are defined by high sphingolipid and cholesterol content, and low buoyant density in high-speed ultracentrifugation gradients (Munro, 2003; Edidin, 2003; Brown, 2000; Simons, 1997). Lipid rafts are now thought to include a variety of plasma membrane domains with different characteristics that invaginate into endocytic vesicles (Mayor, 2004; Helms, 2004; Sharma, 2003; Cheng, 2006).
The uptake and intracellular trafficking of sphingolipids is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, neurodegenerative disease, and inflammation.
Sphingolipid and cholesterol trafficking is altered in the cells of patients with Niemann Pick disease, and a number of other lipid storage diseases where sphingolipids accumulate in late endosomal and lysosomal compartments (Pagano, 2003; Simons, 2000).
Cholesterol and sphingolipids such as ceramide, sphingomyelin, and gangliosides are also thought to be involved in the pathogenesis of Alzheimer's disease (Cutler, 2004; Han, 2005; Mattson, 2005; Soreghan, 2003).
Many viruses and pathogens, including the Alzheimer's associated amyloid peptide, recognize specific carbohydrate headgroups of glycosphingolipids (GSLs) (Sandvig, 2004; Smith, 2004; Yanagisawa, 1995; Mahfoud, 2002), a large variety of which are expressed on the surfaces of cells and occupy lipid raft domains (Degroote, 2004; Simons, 1988).
In spite of keen interest in this field, imaging rafts in living cells has been problematic. Many argue that rafts are extremely difficult to detect in the plasma membrane using standard visible light techniques such as fluorescence widefield or confocal microscopy because of the small size of the rafts, as measured by a variety of quantitative fluorescence techniques (e.g. single particle tracking, FRET, fluorescence anisotropy, (Pralle, Keller et al. 2000; Sharma, Varma et al. 2004; Rao and Mayor 2005)). One explanation for such difficulties in detection may be because rafts are very likely nanoscale dynamic structures, and that they may coalesce into rapidly endocytosing domains (Janes, Ley et al. 1999; Mayor and Rao 2004; Paladino, Sarnataro et al. 2004; Schuck and Simons 2004; Hancock 2006) whose transient nature renders them difficult to identify at the plasma membrane.
Additionally, lipid domains with raft-like characteristics occur in several different cellular organelles, implying that they are not only domains for uptake and transduction by plasma-membrane bound molecules, but rather transport domains for vesicular trafficking between various organelles (van Meer and Lisman 2002; Mayor and Riezman 2004; Schuck and Simons 2004; Paladino, Pocard et al. 2006).
Currently, very little is known about how different ligands associate with raft domains, to what extent lipid content in those domains differs, and what effect raft lipids have on intracellular targeting. It appears possible to answer important questions about the trafficking fate of raft constituents by imaging the endocytic domains that they form. To begin to answer these questions, it is important to develop a diverse battery of markers to characterize the determinants of binding and trafficking behaviours. However, currently available methods to label the trafficking pathways of sphingolipids in live cells are limited.
Raft-associated proteins such as cholera toxin (CTxB), glycosyl phosphatidylinositol (GPI)-anchored proteins and flotillin have been used to study the intracellular itineraries of raft borne proteins and lipids (Glebov, 2006; Sabharanjak, 2002).
Recently, fluorescently conjugated CtxB has been the main label of choice (Invitrogen). However, CtxB appears to recognize only a specific subset of raft domains, as it does not overlap extensively with at least two other raft localized proteins, flotillin and lysenin toxin (Glebov et al, 2006). Additionally, it appears that CtxB uptake is not exclusively raft-mediated.
CtxB and fluorescent sphingolipid analogs both have disadvantages and may disrupt the process of raft-mediated endocytosis itself.
CtxB tends to induce raft clustering (Janes et al, 1999; Schuck and Simons, 2004); as well, CtxB is routinely detected using an antibody. Antibody binding to cell surface ligands is known to lead to clustering of microdomains and their resident proteins, and increased endocytic uptake, and thus, will be expected to perturb the natural trafficking behavior of the sphingolipid rafts to which it binds.
Another series of commercially available sphingolipid markers consist of fluorescently-tagged sphingolipid analogs. These markers can be used in living cells, but their trafficking behavior in cells is distinctly different from endogenous lipids, and have be shown to behave aberrantly due to substitutions of a bulky fluorophore in place of an acyl chain in the lipid. Another disadvantage of these markers is that they necessarily increase the sphingolipid content of the cells to be observed, and therefore could be expected to interfere with normal raft trafficking behavior.
Another group has used lysenin, a protein toxin from earthworm, to label sphingolipid domains. Lysenin is commercially available as a purified 297 amino acid peptide from Sigma-Aldrich, and Peptide Institute, Japan.
Flotillin and lysenin are potentially good markers, but are not easily obtained or externally applied to a cell surface, due to the necessity of either transfecting and translating the protein (flotillin), or using bacterially produced recombinant protein (lysenin). Recent data suggests that lysenin does not associate with biochemically isolated detergent resistant membranes, which may contain lipid raft domain proteins.
A number of groups have used a lipid raft targeting domain long considered standard, GPI (glycosylphosphatidylinositol) fused with green fluorescent protein, known as GPI-GFP. This construct is expressed as a transgene in cells, which carries the signal for covalent attachment of the raft-targeting lipid moiety. However, questions have arisen regarding the faithfulness of lipid raft localization by this marker. It is known that the GPI-GFP can confer different targeting behaviour depending on the particular lipid moiety that is attached (Mayor and Riezman 2004), and that up to ˜70% of the population of GPI-linked proteins are not present in rafts at the membrane (Sharma, Varma et al. 2004). Although (GPI-)GFP has been used in many published reports as a “raft marker”, its role as a bona-fide marker of sphingolipid-rich domains is in doubt, as in most cases it appears to label the plasma membrane uniformly.
Therefore, a sphingolipid-targeted, exogenous probe for live imaging studies would be a useful tool in studying diseases whose pathogenesis is GSL-dependent. To date, there are no available non-invasive, non-transgenic small-molecule probes that can be exogenously applied to cells, for visualising and trafficking of sphingolipid-containing microdomains.